Academic literature on the topic 'Organohalides'

ABSTRACT Organohalides are organic molecules formed biotically and abiotically, both naturally and through industrial production. They are usually toxic and represent a health risk for living organisms, including humans. Bacteria capable of degrading organohalides for growth express dehalogenase genes encoding enzymes that cleave carbon-halogen bonds. Such bacteria are of potential high interest for bioremediation of contaminated sites. Dehalogenase genes are often part of gene clusters that may include regulators, accessory genes and genes for transporters and other enzymes of organohalide degradation pathways. Organohalides and their degradation products affect the activity of regulatory factors, and extensive genome-wide modulation of gene expression helps dehalogenating bacteria to cope with stresses associated with dehalogenation, such as intracellular increase of halides, dehalogenase-dependent acid production, organohalide toxicity and misrouting and bottlenecks in metabolic fluxes. This review focuses on transcriptional regulation of gene clusters for dehalogenation in bacteria, as studied in laboratory experiments and in situ. The diversity in gene content, organization and regulation of such gene clusters is highlighted for representative organohalide-degrading bacteria. Selected examples illustrate a key, overlooked role of regulatory processes, often strain-specific, for efficient dehalogenation and productive growth in presence of organohalides.

Organohalide pollution of subsurface environments is ubiquitous across all industrialised countries. Fortunately, strictly anaerobic microorganisms exist that have evolved using naturally occurring organohalides as their terminal electron acceptor. These unusual organisms are now being utilised to clean anthropogenic organohalide pollution.

AbstractThe unique chemical and physical properties of nanoscale materials have led to important roles in several scientific and technological fields. Environmental chemistry processes have benefited from the enhanced reactivity of nanoscale particles relative to their bulk counterparts with contaminants. Here, we describe recent advances in the synthesis and characterization of metallic and bimetallic nanoparticles that have been effective toward degrading toxic organohalide contaminants. We then review the degradation mechanisms involved in the reactions of nanoscale particles with organohalides via reduction pathways. We also discuss an emerging area – the degradation of organohalides via multi-electron transfer pathways.

In this study, a bioremediation approach was evaluated for the decontamination of an aquifer affected by the release of organohalides by an industrial landfill. After preliminary physicochemical and microbiological characterization of the landfill groundwater, the stimulation of natural organohalide respiration by the addition of a reducing substrate (i.e., molasse) was tested both at microcosm and at field scales, by the placement of an anaerobic permeable reactive bio-barrier. Illumina sequencing of cDNA 16S rRNA gene revealed that organohalide-respiring bacteria of genera Geobacter, Sulfurospirillum, Dehalococcoides, Clostridium and Shewanella were present within the aquifer microbial community, along with fermentative Firmicutes and Parvarchaeota. Microcosm experiments confirmed the presence of an active natural attenuation, which was boosted by the addition of the reducing substrate. Field tests showed that the bio-barrier decreased the concentration of chloroethenes at a rate of 23.74 kg d−1. Monitoring of organohalide respiration biomarkers by qPCR and Illumina sequencing revealed that native microbial populations were involved in the dechlorination process, although their specific role still needs to be clarified. The accumulation of lower-chloroethenes suggested the need of future improvement of the present approach by supporting bacterial vinyl-chloride oxidation, to achieve a complete degradation of chloroethenes.

Halogenated organic matter buried in marine subsurface sediment may serve as a source of electron acceptors for anaerobic respiration of subseafloor microbes. Detection of a diverse array of reductive dehalogenase-homologous ( rdhA ) genes suggests that subseafloor organohalide-respiring microbial communities may play significant ecological roles in the biogeochemical carbon and halogen cycle in the subseafloor biosphere. We report here the spatial distribution of dehalogenation activity in the Nankai Trough plate-subduction zone of the northwest Pacific off the Kii Peninsula of Japan. Incubation experiments with slurries of sediment collected at various depths and locations showed that degradation of several organohalides tested only occurred in the shallow sedimentary basin, down to 4.7 metres below the seafloor, despite detection of rdhA in the deeper sediments. We studied the phylogenetic diversity of the metabolically active microbes in positive enrichment cultures by extracting RNA, and found that Desulfuromonadales bacteria predominate. In addition, for the isolation of genes involved in the dehalogenation reaction, we performed a substrate-induced gene expression screening on DNA extracted from the enrichment cultures. Diverse DNA fragments were obtained and some of them showed best BLAST hit to known organohalide respirers such as Dehalococcoides , whereas no functionally known dehalogenation-related genes such as rdhA were found, indicating the need to improve the molecular approach to assess functional genes for organohalide respiration.

Silylboranes are used as borylation reagents for organohalides in the presence of alkoxy bases without transition-metal catalysts. PhMe2Si–B(pin) reacts with a variety of aryl, alkenyl, and alkyl halides, including sterically hindered examples, to provide the corresponding organoboronates in good yields with high borylation/silylation ratios, showing good functional group compatibility. Halogenophilic attack of a silyl nucleophile on organohalides, and subsequent nucleophilic attack on the boron electrophile are identified to be crucial, based on the results of extensive theoretical and experimental studies. This boryl­ation reaction is further applied to the first direct dimesitylboryl (BMes2) substitution of aryl halides using Ph2MeSi–BMes2 and Na(O-t-Bu), affording aryldimesitylboranes, which are regarded as an important class of compounds for organic materials.1 Introduction2 Boryl Substitution of Organohalides with PhMe2Si–B(pin)/Alkoxy Bases3 Mechanistic Investigations4 DFT Mechanistic Studies Using an Artificial Force Induced Reaction (AFIR) Method5 Dimesitylboryl Substitution of Aryl Halides with Ph2MeSi–BMes2/Na(O-t-Bu)6 Conclusion

A CNDO/2 parameterization for performing semiempirical molecular orbital calculations for organic molecules containing bromine and iodine is presented; the results are superior to those from other parameterizations, and generally agree with ab initio calculations and experiment.

Dehalobacter restrictus strain PER-K23 is an obligate organohalide respiring bacterium, which displays extremely narrow metabolic capabilities. It grows only via coupling energy conservation to anaerobic respiration of tetra- and trichloroethene with hydrogen as sole electron donor. Dehalobacter restrictus represents the paradigmatic member of the genus Dehalobacter , which in recent years has turned out to be a major player in the bioremediation of an increasing number of organohalides, both in situ and in laboratory studies. The recent elucidation of the D. restrictus genome revealed a rather elaborate genome with predicted pathways that were not suspected from its restricted metabolism, such as a complete corrinoid biosynthetic pathway, the Wood–Ljungdahl (WL) pathway for CO 2 fixation, abundant transcriptional regulators and several types of hydrogenases. However, one important feature of the genome is the presence of 25 reductive dehalogenase genes, from which so far only one, pceA , has been characterized on genetic and biochemical levels. This study describes a multi-level functional genomics approach on D. restrictus across three different growth phases. A global proteomic analysis allowed consideration of general metabolic pathways relevant to organohalide respiration, whereas the dedicated genomic and transcriptomic analysis focused on the diversity, composition and expression of genes associated with reductive dehalogenases.

A serious impediment to the application of fundamental toxicology to the protection of human and environmental health is that most organisms are exposed to mixtures of chemical agents while the majority of toxicology research elucidates the toxic actions of individual agents. Our current state of knowledge is insufficient for predicting the effects of a combination of agents based on the dose-response characteristics of the agents administered singly. Even when dose-response data from mixtures and their individual components are available, no clear consensus exists as to which means are appropriate for determining if an interaction such as synergy or antagonism is indicated by those data. Sound mathematical analysis of toxic interaction is an essential ingredient in this pursuit. Experimental designs and means of data analysis permitting precarious conclusions remain in common use, and impede the characterization and elucidation of xenobiotic interactions. This thesis critiques some of the approaches used to address xenobiotic interaction, and offers specific and novel techniques and guidelines for improved approaches. Increasingly large numbers of toxicants exceed our current ability to assess toxicity. The development of in vitro methods offers an increased ability to examine larger numbers of toxicants and their combinations than conventional in vivo approaches given the finite resources available. This thesis presents evidence supporting the validation of precision-cut liver slice culture as an in vitro model for investigating hepatotoxic interactions of defined binary mixtures. Toxic interactions observed in vivo were demonstrated in the in vitro liver slice culture in two strains of rat. No intrinsic bias was detected by challenging this approach with a sham interaction (one compound combined with itself). Structure-activity based predictions of toxic interaction were demonstrated in liver slice culture. Two separate means of data analysis arrived at the same interpretations of the data for all of the experimental results described above. No toxic interactions were found in a limited but rigorous test of a bacterial toxicity assay, suggesting that interactive toxic responses are sensitive to the choice of biological model. Preliminary experiments were conducted for assessing the effect of mechanistic probes (metabolic manipulations) on established toxic interactions.

Les halogènes présentent des propriétés très intéressantes en chimie organique. L’incorporation de ceux-ci sur des molécules organiques permet de modifier leurs propriétés et les rend indispensables dans plusieurs domaines tels que la chimie pharmaceutique, l’agrochimie et la science des matériaux. En plus de cela, ils sont de très bons intermédiaires de synthèse pour l’obtention de produits hautement fonctionnalisés. Cette thèse portera donc sur le développement de nouvelles méthodes pour l’incorporation d’halogènes à partir de produits de départ simples. Dans le premier projet, une réaction d’hydrofluoration a été développée. Cela permet d’incorporer un atome de fluor sur diverses molécules, à partir d’alcènes qui sont facilement accessibles. Les conditions réactionnelles sont une combinaison entre un acide fort, l’acide méthanesulfonique et une source de fluorure, Et3N·3HF. Les conditions ont pu être appliquées à des alcènes comportant une vaste gamme de groupements fonctionnels. La réaction n’est toutefois compatible qu’avec les alcènes 1,1-disubstitués et trisubstitués. Les rendements obtenus sont généralement bons et comparables à ceux obtenus avec les autres méthodes de la littérature. Dans le deuxième projet, une modification des conditions réactionnelles du premier projet a été réalisée pour permettre l’hydrochloration, l’hydrobromation et l’hydroiodation d’alcènes. Les composés halogénés sont obtenus dans d’excellents rendements, généralement sans purification. Des études mécanistiques ont été effectuées et montre que la réaction est favorisée par la formation d’un intermédiaire acétate, qui provient de la réaction entre le substrat et le solvant, l’acide acétique. Enfin, un exemple de deutérochloration a également été montré. Pour ce qui est du troisième projet, nous nous sommes intéressés à la déoxyfluoration d’alcools. L’objectif était de complémenter les méthodes présentes dans la littérature qui permettent la transformation sur des alcools primaires et secondaires, mais qui sont souvent problématiques pour les alcools tertiaires. En modifiant les conditions réactionnelles du premier projet, nous avons été en mesure de développer une réaction de déoxyfluoration d’alcools tertiaires qui donne d’excellents rendements. La réaction est compatible avec une vaste gamme de groupements fonctionnels, et les produits fluorés correspondants sont obtenus généralement sans purification. Une extension de la méthode pour d’autres types de liaison C–O a aussi été effectuée. Enfin, des études mécanistiques ont été réalisées et ont permis de déterminer que la réaction se déroule via une séquence d’élimination puis d’hydrofluoration. Une modification des conditions réactionnelles de déoxyfluoration a également été effectuée pour les rendre compatibles avec les méthodes de radiofluoration
Halides have very interesting properties in organic chemistry. Organic molecules that bear halides have modified properties which make them interesting in many fields of chemistry such as pharmaceuticals, agrochemicals, and material science. Moreover, they are useful intermediate for the synthesis of a vast array of highly functionalized molecules. This thesis will therefore report on the development of new reaction for the incorporation of halides starting from abundant molecules such as alkenes and alcohols. The first project focused on the development of an hydrofluorination reaction. This allowed for the easy incorporation of a fluorine atom on various molecules starting from easily available alkenes. The reaction uses a combination of a strong acid, methanesulfonic acid, and a fluoride source, triethylamine trihydrofluoride. These conditions were compatible with a wide range of functional groups. However, the reaction was limited to 1,1-disubstituted and trisubstituted alkenes. The yields are generally good and similar to those of other reported methods. For the second project, a modification of the reaction conditions from the first project was performed to allow for the hydrochlorination, hydrobromination and hydroiodination of alkenes. The corresponding halides are obtained in excellent yields, usually without purification. Mechanistic studies have shown that the solvent, acetic acid, plays a role in the stabilization of the carbocation. Finally, an example of deuteriochlorination has been reported using deuterated acetic acid. In the third project, we focused on the transformation of alcohols into fluorides. The main objective of this project was to complement the existing methods of deoxyfluorination which work generally well on primary and secondary alcohols, but not so much on tertiary alcohols. By modifying the conditions from the first project, we were able to develop a deoxyfluorination reaction that gives tertiary fluorides in excellent yields. The reaction is compatible with a vast array of functional groups and the products are usually obtained without purification. The reaction has been extended for the fluorination of other C–O bonds such as ethers and esters. Mechanistic studies have been performed and show that the reaction proceeds in two steps via an elimination/hydrofluorination pathway. Finally, a modification of these conditions has been done to allow for an adaptation of this reaction in radiofluorination of alcohols

Solar cells based on organohalide perovskites (PSCs) have made rapid progress in recent years and are a promising emerging technology. An important next evolutionary step for PSCs is their up-scaling to commercially relevant dimensions. The main challenges in scaling PSCs to be compatible with current c-Si cells are related to the limited conductivity of the transparent electrode, and the processing of a uniform and defect-free organohalide perovskite layer over large areas. In this work we present a generic and simple approach to realizing efficient solution-processed, monolithic solar cells based on methylammonium lead iodide (CH₃NH₃PbI₃). Our devices have an aperture area of 25 cm² without relying on an interconnected strip design, therefore reducing the complexity of the fabrication process and enhancing compatibility with the c-Si cell geometry. We utilize simple aluminum grid lines to increase the conductivity of the transparent electrode. These grid lines were exposed to an UV-ozone plasma to grow a thin aluminum oxide layer. This dramatically improves the wetting and film forming of the organohalide perovskite junction on top of the lines, reducing the probability of short circuits between the grid and the top electrode. The best devices employing these modified grids achieved power conversion efficiencies of up to 6.8%.

La freqüent contaminació d’aigua subterrània per compostos organohalogenats és un greu problema degut als riscs humans i ecològics que se’n deriven. La bioremediació és un tècnica sostenible que permet superar algunes de les limitacions que presenten els tractaments fisicoquímics. En aquest estudi ens proposem obtenir i caracteritzar cultius que contenen bacteris anaerobis capaços de degradar compostos organohalogenats ambientalment perillosos i que es puguin aplicar per a la bioremediació d’aqüífers in situ. En treballs previs realitzats al nostre laboratori es va obtenir un cultiu enriquit que contenia un bacteri dehalorespirador del gènere Dehalogenimonas a partir de sediments de la desembocadura del riu Besòs (Barcelona) que degrada alcans amb halògens situats en carbons adjacents. En aquesta tesis, s’ha identificat la dehalogenasa reductora (RDasa) d’aquesta Dehalogenimonas implicada en la conversió de dibromur d’etilè (EDB) al compost innocu etilè combinant tècniques de proteòmica basades en gels d’electroforesis, tests enzimàtics i nano-cromatografia líquida acoblada a espectrometria de masses (nLC-MS/MS). Aquesta RDasa es va designar com a EdbA. EdbA és la primera RDasa identificada entre les espècies d’aquest gènere bacterià que catalitza una reacció de debromació. A més, és la primera RDasa que s’ha demostrat funcional i que no té cap subunitat B de fixació a la membrana citoplasmàtica codificada de forma adjacent en el seu genoma. Addicionalment, s’ha detectat un enzim ortolog a l’enzim responsable de la degradació de 1,2-diclorpropà a propé (DcpA) com a única RDasa en cultius que transformen 1,2,3-triclorpropà a clorur d’alil mitjançant la combinació de tècniques d’ultracentrifugació, gels d’electroforesis i nLC-MS/MS. Aquesta DcpA es va detectar en la fracció de la membrana tal i com predeien les eines bioinformàtiques emprades. El mecanisme pel qual aquestes dues RDases identificades es fixen a les membranes és encara desconegut. En aquesta treball s’ha obtingut un segon consorci bacterià estable provinent de llots d’una planta de tractament d’aigües residuals industrials i aplicant estratègies de d’enriquiment del cultiu i tècniques de dilució fins a l’extinció. Aquest cultiu fermenta diclorometà (DCM) i dibromometà (DBM) en acetat i format. S’ha demostrat que el bacteri responsable de la fermentació d’aquests dihalometans és un Dehalobacterium i s’ha procedit al seu aïllament. Tanmateix, les interaccions sinèrgiques entre les espècies del consorci han impedit el seu aïllament. Mitjançant la selecció de colònies en cultius semi sòlids, canvis en la composició del medi i l’ús de antibiòtics, s’ha assolit un cultiu on l’abundància de Dehalobacterium és del 67%. L´acompanyen bacteris dels gèneres Acetobacterium i Desulfovibrio, tal i com revelen els anàlisis de genoteques. El fraccionament dels isòtops de carboni durant la fermentació de DCM per aquest cultiu s’ha determinat mitjançant l’anàlisi d’isòtops estables de compostos específics (CSIA). El valor obtingut de -27 ± 2‰ difereix del prèviament publicat per una soca de Dehalobacter (-15.5 ± 1.5‰) que també fermentava DCM. Aquests valors són significativament diferent dels obtinguts per bacteris metilotròfics degradadors de DCM (que varien de -45 a -61‰) i podria permetre la distinció entre vies de degradació de DCM en treballs de bioremediació in situ. Finalment, s’ha demostrat que la presència de co-contaminants que es detecten freqüentment amb DCM, tals com tricloroetilè (TCE), 1,2-dicloroetà (1,2-DCA), cis-dicloroetilè (cis-DCE), 1,1,2-tricloroetà (1,1,2-TCA), àcid perfluorooctanoic (PFOA) i 3,4-dicloroanilina (3,4-DCA) no provoca una inhibició significativa en la degradació de DCM pel cultiu amb Dehalobacterium a les concentracions testades. La concentració de cloroform de 100 mg/L provoca una total inhibició. De manera similar, la presència de 200 mg/L d’àcid perfluorooctanosulfonic (PFOS) i ≥ 25 mg/L de diuron provoquen una inhibició severa, impedint la degradació completa de DCM. Tanmateix, l’activitat degradadora de DCM es recupera quan els cultius inhibits es transfereixen a medi fresc sense co-contaminants.
La frecuente contaminación de las aguas subterráneas por compuestos organohalogenados es un grave problema ambiental debido a los riesgos ecológicos y para la salud humana de ella derivados. La bioremediación es una tecnología sostenible que evita algunos inconvenientes que presentan los tratamientos físico-químicos. En este estudio nos proponemos obtener y caracterizar cultivos que contengan bacterias anaerobias que degraden compuestos organohalogenados ambientalmente peligrosos con potencial para la bioremediación in situ de aguas subterráneas. En trabajos previos de nuestro grupo de investigación, se obtuvo un cultivo enriquecido en bacterias del género Dehalogenimonas procedente de sedimentos del estuario del río Besós (Barcelona) que degrada alcanos con halógenos situados en carbonos adyacentes. En esta tesis se ha identificado la dehalogenasa reductora (RDasa) de esta cepa de Dehalogenimonas implicada en la conversión del dibromuro de etileno (EDB) al compuesto inocuo eteno combinando técnicas de proteómica basadas en geles de electroforesis, ensayos enzimáticos y nano-cromatografía líquida de alta resolución (nLC-MS/MS). Esta RDasa es designada EdbA, y constituye la primera RDasa identificada en este género bacteriano que cataliza una reacción de debromación. Además, es también la primera RDasa en ser demostrada funcional sin una subunidad B de anclaje a la membrana codificada de forma adyacente en el genoma. Adicionalmente, se ha detectado una única RDasa en cultivos que transforman 1,2,3-tricloropropano a cloruro de alilo combinando técnicas de ultracentrifugación, geles de electroforesis y nLC-MS/MS. Esta enzima ortóloga a DcpA, la responsable de la degradación de 1,2-dicloropropano a propeno, ha sido detectada en la fracción proteica de membrana, lo cual concuerta con las predicciones realizadas mediante herramientas bioinformáticas. El mecanismo por el cual EdbA y esta DcpA se anclan a la membrana citoplasmática es desconocido, atribuyéndose a proteínas todavía no descritas. En este trabajo se ha obtenido un segundo consorcio bacteriano estable a partir de lodos de una planta de tratamiento de aguas residuales industriales aplicando técnicas de cultivo de enriquecimiento y dilución por extinción. Este cultivo fermenta diclorometano (DCM) y dibromometano (DBM) a acetato y formato. Se ha demostrado que la bacteria responsable de la fermentación pertenece al género Dehalobacterium, y se ha procedido a su aislamiento. Sin embargo, las interacciones sinérgicas existentes entre las especies del consorcio han impedido obtener un cultivo puro. Seleccionando colonias en medio de cultivo semisólido, aplicando antibióticos y cambios en la composición del medio, se ha obtenido una abundancia relativa de Dehalobacterium del 67%. Le acompañan bacterias de los géneros Acetobacterium y Desulfovibrio, tal y como se detectó mediante análisis de genotecas. El fraccionamiento isotópico del carbono durante la fermentación del DCM por este cultivo fue determinado mediante análisis de isótopos estables de compuestos específicos (CSIA). El valor obtenido, -27 ± 2‰, difiere del publicado previamente para una cepa de Dehalobacter que también fermenta el DCM (-15.5 ± 1.5‰). Estos valores son significativamente diferentes de los obtenidos con bacterias metilotróficas degradadoras de DCM (-45 a -61‰), y podrían permitir diferenciar vías de degradación de DCM en trabajos de bioremediación in situ. Finalmente, se ha demostrado que la presencia de co-contaminantes que se detectan frecuentemente con el DCM, como el tricloroetileno (TCE), 1,2-dicloroetano (1,2-DCA), cis-dicloroetileno (cis-DCE), 1,1,2-tricloroetano (1,1,2-TCA), ácido perfluorooctanoico (PFOA) y 3,4-dicloroanilina (3,4-DCA) no provocan una inhibición significativa en la degradación de DCM por parte del cultivo de Dehalobacterium, a las concentraciones estudiadas. Una concentración de cloroformo de 100 mg/L provoca una inhibición total. De manera similar, 200 mg/L de sulfonato de perfluoroctano (PFOS), y ≥ 25 mg/L de diuron provocan una inhibición severa, impidiendo la degradación completa del DCM. Sin embargo, la actividad degradadora de DCM se recupera cuando los cultivos inhibidos se transfieren a medio libre de co-contaminantes.
The widespread groundwater contamination by organohalide compounds is of a major concern due to the human and ecological risks derived from it. Bioremediation is a sustainable technology that overcomes some limitations of the physical-chemical remediation techniques on these water bodies. In this study, we aimed to obtain and characterize cultures containing anaerobic bacteria capable of degrading organohalide compounds of environmental concern with potential for in situ groundwater bioremediation. In previous work carried out in our laboratory a highly enriched culture containing organohalide-respiring bacteria from the genus Dehalogenimonas degrading vicinally halogenated alkanes was obtained from sediments of the river Besós estuary (Barcelona). In this thesis, the reductive dehalogenase (RDase) from this Dehalogenimonas strain responsible for the catalysis of ethylene dibromide (EDB) to the innocuous ethene was identified combining gel-based proteomic techniques, specific enzymatic tests and nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS). This RDase is therefore designated as EdbA, for ethylene dibromide RDase subunit A. EdbA is the first RDase identified for debrominating catalytic activity among species of this genus. Moreover, it is the first RDase shown to be functional for respiration without an adjacent membrane-anchoring subunit B encoded on the genome. Additionally, combining ultracentrifugation, gel electrophoresis and nLC-MS/MS, an orthologous enzyme of the dichloropropane-to-propene RDase (DcpA) was the only RDase detected in 1,2,3-trichloropropane-to-allyl chloride dehalogenating cultures. This DcpA was detected in the membrane fraction of the crude protein extract, in accordance to its predicted subcellular localization by bioinformatics tools and it is also not co-localised with an rdhB gene. The membrane-anchoring mechanisms of these RDases remains not known and may rely in yet-unidentified proteins. A second stable bacterial consortium was obtained in the present work from slurry samples of an industrial wastewater treatment plant with a combination of enrichment culture strategies and the dilution-to-extinction technique. This culture was demonstrated to ferment dichloromethane (DCM) and dibromomethane (DBM) into acetate and formate. The Dehalobacterium sp. present in this culture was shown to be the responsible for the dihalomethanes fermentation, and the isolation of this strain was attempted. However, the synergic interactions existing among the different accompanying species present in the bacterial consortia impeded the isolation. Despite a pure culture was not achieved via picking up colonies from semisolid agar cultures, changes in the medium composition, and the application of selected antibiotics, a final relative abundance of Dehalobacterium sp. of 67 % was attained. As determined by clone library analysis, bacteria from the genera Acetobacterium and Desulfovibrio remained present in the culture. The carbon isotope fractionation during DCM fermentation by this culture was determined by compound-specific stable isotope analysis (CSIA). The value obtained was -27 ± 2‰ and differs from the previously published value of -15.5 ± 1.5‰ of a Dehalobacter sp. performing also DCM fermentation. These values are yet significantly different from those reported for facultative methylotrophic bacteria degrading DCM (ranging from -45 to -61‰), and this would allow for further differentiation of these degradation pathways during in situ bioremediation works. Finally, the potential inhibitory effect of selected frequent groundwater co-contaminants over DCM degradation by the Dehalobacterium-containing culture was assessed for further in situ bioremediation applications. Trichloroethylene (TCE), 1,2-dichloroethane (1,2-DCA), cis-dichloroethylene (cis-DCE), 1,1,2-trichloroethane (1,1,2-TCA), perfluorooctanoic acid (PFOA), and 3,4-dichloroaniline (3,4-DCA) did not show significant inhibitory effects at the concentrations tested. Differently, a total inhibition was caused with a chloroform concentration of 100 mg/L. Also, the presence of 200 mg/L of perfluorooctanesulfonic acid (PFOS), as well as concentrations higher than 25 mg/L of the pesticide diuron caused a severe inhibitory effect, preventing the full depletion of DCM. Nevertheless, DCM degrading activity was recovered when inhibited cultures were transferred to co-contaminant free medium.

Organohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.

Microbial respiration can be highly diverse and adaptable, with many bacteria able to respond to changes in their environment promptly and efficiently. The regulation of respiratory enzymes by highly responsive and precise transcriptional regulators confers distinct advantage for survival in sometimes harsh and extreme conditions. The organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2 is able to utilise a wide range of electron acceptors and respiratory processes through tight regulation of respiratory machinery. An example of this tight regulation of respiratory machinery can been seen by biochemical analysis of the CRP-FNR-type transcriptional regulator family CprK, of which five are present in the strain. CprK1 is able to sense the presence of the physiological ligand, 3-chloro-4-hydroxyphenylacetic acid (CHPA), of reductive dehalogenase CprA1 with nM affinity. In this work we demonstrate that CprK1 is able to distinguish between the chlorinated CprA1 substrate CHPA and the non-chlorinated product 4-hydroxyphenylacetic acid (HPA) by ‘pKa interrogation’ of the 4-hydroxy moiety and by the atomic radius of the ortho-moiety. Through the use of in vitro biophysical and in vivo transcriptional response assays, we show that CprK1 is able to sense a number of halogenated phenols, including phenylacetic acids and nitrophenols. We also demonstrate that a 4-hydroxyl group is essential for CprK1 activation. In Chapter 4, an attempt to modify the effector sensitivity of CprK1 is performed by site-specific and random mutagenesis, and mutant selection assays are developed. We show that CprK1 is highly resistant to effector specificity modifications, with seemingly minor or conservative amino acid changes removing CprK1’s ability to initiate transcription. In Chapter 5, the CprK1 paralogue, CprK4 from D. hafniense DCB-2 is characterised by in vitro biophysical and in vivo transcriptional response assays in order to assess its potential as a biosensor. We show that CprK4 is able to bind cis-regulatory DNA elements dehaloboxes 7 and 10 in the absence of effector by Surface Plasmon Resonance (SPR) protein array; however, we were unable to identify its effectors reliably. Due to the unknown nature of CprK4’s effector, it is still unclear whether CprK4 could be a valuable biosensor.

The evolution of microorganisms over millions of years has led to an impressive adaptability regarding the utilisation of different environmental conditions. The identification of bacterial species with the fascinating features to use cobalamin-dependent metalloenzymes to (i) extract energy from halogenated organic compounds (organohalides) and (ii) transfer methyl groups from lignin breakdowns into central carbon pathways, are examples for this adaptability. The biochemical study of these two cobalamin-dependent enyzmes is the topic of this PhD project. For the extraction of growth energy, organohalides serve as terminal electron acceptorsand are reductively dehalogenated in a respiratory manner termed organohalide respiration. Reductive dehalogenases, the key enzymes in organohalide respiration, catalyse the chemical cleavage between the halogen substituent and the carbon moiety. They use cobalamin and two Fe-S clusters as cofactors and constitute a new and distinct class of cobalamin-dependent enzymes. Their three-dimensional structure and the mechanism of catalysis are unknown, because their hydrophobicity and oxygen sensitivity have hampered their biochemical investigation. Here, a novel purification technology in Escherichia coli for the reductive dehalogenase PceA from Dehalobacter restrictus has been developed, accompanied by methods that allow the in vitro reconstitution of PceA with both cofactors, cobalamin and Fe-S clusters. It has been demonstrated that the soluble expression of PceA is dependent on the covalent fusion of the enzyme to a trigger factor chaperone. Based on these findings, the PceA specific trigger factor PceT has been studied biochemically, resulting in its successful crystallisation. The established protocols for PceA and PceT are transferable to other members of their respective families, which will therefore allow detailed studies of reductive dehalogenases and their associated chaperones in the future. In addition to reductive dehalogenases, organohalide respiring bacteria contain anothercobalamin-dependent enzyme system, termed O-demethylase, which is involved in the carbon metabolism of different anaerobic bacteria. O-demethylases are three-component enzyme systems that transfer methyl groups from aromatic methyl ethers totetrahydrofolate via methylcobalamin intermediates. The different cofactors (substrate,cobalamin and tetrahydrofolate), bind to either of the three individual proteins involvedin O-demethylation. It has been speculated that the same or similar halogenated aromatic molecules are substrates for both organohalide respiration and O-demethylation in the same bacteria. In order to test this proposal, a O-demethylase from Desulfitobacterium hafniense DCB-2 has been studied using X-ray crystallography and biochemistry. As a result, the first crystal structures of the cobalamin-binding protein in complex with cobalamin, and of the methyl acceptor protein in complex with substrate (tetrahydrofolate) and product (methyltetrahydrofolate) from a O-demethylase have been solved toresolutions of 1.5 A, 1.8 A and 1.6 A, respectively. The crystal structures, in combinationwith spectroscopic and biophysical analyses, have led to a proposed mechanism forthe catalysed methyl transfer reaction from methylcobalamin to tetrahydrofolate.

This dissertation is devoted to the development of novel devices for optoelectronic and photovoltaic applications using the promise of inkjet printing with two-dimensional (2D) materials. A systematic approach toward the characterization of the liquid exfoliated 2D inks comprising of graphene, molybdenum disulfide (MoS2), tungsten diselenide (WSe2), and 2D perovskites is discussed at depth. In the first study, the biocompatibility of 2D materials -- graphene and MoS2 -- that were drop cast onto flexible PET and polyimide substrates using mouse embryonic fibroblast (STO) and human esophageal fibroblast (HEF) cell lines, was explored. The polyimide samples for both STO and HEF showed high biocompatibility with a cell survival rate of up to ~ 98% and a confluence rate of 70-98%. An inkjet printed, biocompatible, heterostructure photodetector was constructed using inks of photo-active MoS2 and electrically conducting graphene, which facilitated charge collection of the photocarriers. The importance of such devices stems from their potential utility in age-related-macular degeneration (AMD), which is a condition where the photosensitive retinal tissue degrades with aging, eventually compromising vision. The biocompatible inkjet printed 2D heterojunction devices were photoresponsive to broadband incoming radiation in the visible regime, and the photocurrent scaled proportionally with the incident light intensity, exhibiting a photoresponsivity R ~ 0.30 A/W. Strain-dependent measurements were also conducted with bending, that showed Iph ~ 1.16 µA with strain levels for curvature up to ~ 0.262 cm-1, indicating the feasibility of such devices for large format arrays printed on flexible substrates. Alongside the optoelectronic measurements, temperature-dependent (~ 80 K to 573 K) frequency shifts of the Raman-active E12g and A1g modes of multilayer MoS2 exhibited a red-shift with increasing temperature, where the temperature coefficients for the E12g and A1g modes were determined to be ~ - 0.016 cm-1/K and ~ - 0.014 cm-1/K, respectively. The phonon lifetime τ was determined to be in the picosecond range for the E12g and A1g modes, respectively, for the liquid exfoliated multilayer MoS2. Secondly, an all inkjet printed WSe2-graphene hetero-structure photodetector on flexible polyimide substrates is also studied, where the device performance was found to be superior compared to the MoS2-graphene photodetector. The printed photodetector was photo responsive to broadband incoming radiation in the visible regime, where the photo responsivity R ~ 0.7 A/W and conductivity σ ~ 2.3 × 10-1 S/m were achieved at room temperature. Thirdly, the synthesis of solution-processed 2D layered organo-halide (CH3(CH2)3NH3)2(CH3NH3)n-1PbnI3n+1 (n = 2, 3, and 4) perovskites is presented here, where inkjet printing was used to fabricate heterostructure flexible photodetector devices on polyimide substrates. The ON/OFF ratio was determined to be high, ~ 2.3 × 103 while the photoresponse time on the rising and falling edges was measured to be rise ~ 24 ms and fall ~ 65 ms, respectively. The strain-dependent measurements, conducted here for the first time for inkjet printed perovskite photodetectors, revealed the Ip decreased by only ~ 27% with bending (radius of curvature of ~ 0.262 cm-1). This work demonstrates the tremendous potential of the inkjet printed, composition tunable, organo-halide 2D perovskite heterostructures for high-performance photodetectors, where the techniques are readily translatable toward flexible solar cell platforms as well. Fourthly, metal contacts and carrier transport in 2D (CH3(CH2)3NH3)2(CH3NH3)n-1PbnI3n+1 (n = 4) perovskites is a critical topic, where the use of silver (Ag) and graphene (Gr) inks as metallic contacts to 2D perovskites was investigated. The all inkjet printed Gr-perovskite and Ag-perovskite photodetectors were found to be photo-responsive to broadband incoming radiation where measurements were conducted from λ ~ 400 nm to 2300 nm. The photoresponsivity R and detectivity D were compared between the Gr-perovskite and Ag-perovskite photodetectors, which revealed the higher performance for the Ag-perovskite photodetector. The superior performance of the Ag-perovskite photodetector was also justified with the Schottky barrier analysis using the thermionic emission model through temperature-dependent transport measurements. Finally, this dissertation ends with the description of the first steps for using solution-processed, inkjet printed perovskites for solar cells. The preliminary investigations include the discussion of the chemical formulations for the carrier separation layers, dispersion route, and the variation of solar cell figures of merit with processing.

Organohalide pollution of freshwater and marine sediments threatens human and environmental well-being. In freshwater and marine sediments a natural anaerobic microbiological process called reductive dehalogenation (RD) is carried out by organohalide respiring bacteria (OHRB) and reduces toxicity and improves biodegradability of organohalide pollutants. The reaction is catalyzed by enzymes called reductive dehalogenases (rdh). The marine sediment is the final sink for such dangerous and persistent contaminants, which make the very environment noxious, bioaccumulate in living beings and reach human foodstocks. This work explores reductive dehalogenation, OHRB and their rds’s in the marine environment of two sites of the Adriatic Sea: the Venice Lagoon (VL) and Ravenna Harbor (RH). In a microcosm study, primary sediment from the two sites and from an OHRB-enriched PCB-dechlorinating slurry culture were spiked with different organochloride compounds: hexachlorobenzene (HCBe), 1,2,3,5-tetrachlorobenzene (TeCBe), pentachlorophenol (PCP), 2,3,5-trichlorophenol (TCP) and trichloroethylene (TCE), 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) and a commercial mixture of PCBs (Aroclor© 1254). All compounds were dechlorinated to a certain extent in OHRB-enriched cultures, while primary sediments showed MRD of HCBe, TeCBe, TCP, TCE but not of PCP, and TeCDD, while PCBs dechlorination in VL sediments was not observed in RH but only in VL in previous studies. Microbial community analysis revealed the enrichment of bacteria from the Dehalococcoidia class where dechlorination was taking place in most primary cultures. In enriched cultures the increase of previously identified phylotypes from the same taxon, VLD-1 and VLD-2, correlated with dechlorination. A sediment free TCE-dechlorinating consortium was established in a defined mineral medium. PCR degenerate primer pairs screens and Next Generation Sequencing of amplicons revealed 81 novel rdh homologous gene sequences. Gene expression and proteomic mass spectrometry studies on the TCE-dechlorinating cultures revealed the overexpression of a cluster of three rdh genes hinting at a TCE-dechlorinating activity of one or more of them.